XENOBIOTICA,
1990, VOL. 20,
NO.
7, 671-681
Metabolism of illudin S, a toxic principle of Lampteromycesjaponicus, by rat liver. I. Isolation and identification of cyclopropane ring-cleavage metabolites
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K. TANAKAT, T. INOUET, S. KADOTAS and T. KIKUCHIS
t National Research Institute of Police Science, 6, Sanban-cho, Chiyoda-ku, Tokyo 102, Japan # Research Institute for Wakan-Yaku (Oriental Medicines), Toyama Medical and Pharmaceutical University, 2630, Sugitani, Toyama 930-01, Japan Received 14 September 1989; accepted 2 March 1990
1. Illudin S, a toxic principle of the basidiomycete Lmnpteromyces japonzcuc, was incubated with rat liver 9OOOg supernatant and its metabolites studied. 2. Two metabolites, M1 and M2, were isolated and identified as cyclopropane ringcleavage compounds by n.m.r., i.r. and mass spectral analyses. Moreover, M2 contained a chlorine atom. 3. On the basis of detailed analyses of the 2D n.m.r. spectra and differential nuclear Overhauser effect experiments, the previous assignments of the cyclopropane carbons of illudin S were revised.
Introduction The basidiomycete Lampteromyces japonicus (Japanese name: Tsukiyo-Take) is distinctive in being bioluminescent and is one of the most notorious poisonous mushrooms in Japan. There have been reports of severe illness caused by this mushroom, because occasionally it is eaten by mistake due to its resemblance to edible mushrooms. Chemical investigation of the constituents of this mushroom have been undertaken by several groups of authors (Fukuda et al. 1975, Tsuda et al. 1986) and the isolation of a toxic substance, illudin S (I), having a novel structure with a cyclopropane ring (figure l),has been reported (Nakanishi et al. 1963a, Tada et al. 1964, Nakanishi et al. 1964,1965,Matsumoto et al. 1965). In addition, illudin S has been reported to have antibacterial and antiturnour properties (Itano 1978, Shinozawa et al. 1979, Kelner et al. 1987). Cyclopropane-ring-containing compounds are currently of increasing interest and are used in enzymology, toxicology, etc., and the bioorganic chemistry of cyclopropanes has been explored extensively. It has been demonstrated that in many cases ring-opened metabolites are produced by enzymic oxidation (Suckling 1988). However, no report on the metabolism of illudin. S has yet been published, although knowledge of the metabolic pathways of illudin S in animals and of any biological activities of the metabolites, would be of considerable interest. This paper therefore describes the isolation and identification of illudin S metabolites formed by rat liver 9000g supernatant.
Materials and methods Chicals Illudin S was isolated according to published methods (Nakanishi et al. 1963 a, b, 1964, 1965), from Lampterornyces japomcuc collected in Nagano prefecture in October 1987. 0049-8254/90 $3.00 0 1990 Taylor & Francis Ltd.
K. Tanaka et al.
672
illudin S ( I )
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1
I
OH
M1 (11)
M2(III)
Figure 1. Proposed pathways of metabolism of illudin S by rat liver 9OOOg supernatant, Postulated intermediate shown in parentheses. Illudin S (I): m.p. 125-126"C, [&+ 15lo(c=0.1, methanol), U.V. A M x n m (log 8): 2340 (409), 3180 (3.51);i.r.vmaxcm-': 3408,1693,1659,1600. 'H-and13C-N.m.r.:tables1 and2.Massspectrumm/a: 264 (M'), 233,216,201,189,173. Identity was confirmed by comparing the physical and spectral data with those published. NADP was purchased from Sigma Chemical Co. Ltd (St Louis,MO, USA). All other chemicals and reagents were of the highest grade. Preparation of 9000g supernatant Wistar male rats weighing 150-200 g were obtained from Nippon Bio-Supp. Center (Tokyo, Japan), and allowed free access to standard laboratory chow (MF, Oriental Yeast Co., Tokyo, Japan) and tap water. Rats were killed by decapitation, and livers were quickly removed and chilled in isotonic KCI. A homogenate was prepared by hand, using a glass homogenizer with a Teflon pestle using 2 ml of isotonic KCI per 1 g of liver. A 9OOOg supernatant fraction was prepared by centrifugation of the homogenate for 20min in a Marusan 50-B refrigerated centrifuge. All procedures were carried out at 0 4 ° C . The 9000g supernatant was used fresh on its day of preparation. Incubation A solution of illudin S ( 2 5 0 p o l ) in ethanol ( 2 5 0 ~ 1was ) added to a mixture of NADP (SOpmol), MgCI2(5mmol),and50mlof0.4~phosphatebuffer(pH7.4).Tothismixturewasadded 100mlof9000g supernatant and water to 250 ml. The mixture was incubated for 0.5 h at 37"C, with shaking. For a timecourse study, incubation mixtures consisted of 0 4 m l of 9000gsupernatant fraction, 2.5 pmol of illudin S,
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673
Table 1. 'H-N.m.r. data for illudin S, metabolites M1 and M2 (ppm from tetramethylsilane) Compound Illudin S'
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Proton 472 s 645 s
6-H 8-H 8-H 10-H 11-H 11-H 12-H 12-H 13-H 14-H 15-H 15-H
Mlb 455 s
1-37s 0.42 ddd (97,67,41) 1.12ddd(9.9,6.1,4.1) 0.84 ddd (9.9, 67, 49) @96ddd(97,6*1,49) 1.69 s 1.20s 3.47d(ll) 3.55 d(l1)
MZb
2.44 d (16.2) 2.57 d (162) 210s 273 t (82)
4.57 s 245 d (159) 2-59d (15.9) 211 8 3-02t (98)
3.37 t (82)
3-59t (98)
219s 1-05s 3-01d(10.4) 3*07d(10.4)
221 8 1.04s 3.01 d(10.4) 3.07 d (10.4)
Spectra were measured in CDCI,' and DMSO-d,b solutions. Values in parentheses are coupling constants (Hz). Table 2.
',C-N.m.r. data for illudin S, metabolites M1 and M2 Compound ~
Carbon atom 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Illudin S' 20050(s) 7615 ( 8 ) 3190 (s) 13504( 8 ) 13852 (s) 7484 (d) 551O(s) 141.57(d) 136.06 (s) 2480 (9) 876 (t) 6.04 (t) 1416(q) 15.83 (9) 68.94 (t)
Mlb 148.65 ( 8 ) 122.29 (s) 13425 ( 8 ) 124.37 (s) 14207(s) 7689 (d) 47.58 ( 8 ) 37.60 (t) 12604(s) 11-97(9) 33-44(t) 6Q16(t) 1455 (4) 1689(9) 6767 (t)
M2b 148.82 ( 8 ) 12244 ( 8 ) 133*34(s) 12450 (s) 142-38(s) 7681 (d) 47.58 ( 8 ) 3759 (t) 127-05(s) 11.91 (9) 33*00(t) 42-84(t) 14.46 (9) 1861 (9) 67-58(t)
Spectra were measured in CDCI,' and DMSO-d,'solutions. The multiplicities of carbon signals are indicated as s, d, t, and q. 1pmol of NADP, 100pnol of MgCI,, 400pnol of phosphate buffer (pH 7.4), and water to a final volume of 5mI. Analytical methodc T.1.c. was carried out on Merck Kieselgel60 F ,,, (0.25 mm) platea using (a) ethyl acetatebenzene (6 :4, v/v) or (b) chloroform-methanol(9 :1, v/v) as developing solvents. Spots were detected under U.V. light (254nm) or from coloration by spraying with anisaldehyd-ulphuric acid-ethanol(1: 1 :19,by vol.) reagent and heating at 120°C for 5min. T.1.c. densitometrywas carried out on a Shmadzu CS-9OOO scanner, with the sample beam at 225 nm and zig-zag scanning. Melting points were determined with a Kofler-type apparatus and are uncorrected. U.V.spectra were taken in ethanol solutions and i.r. spectra in KBr discs. N.m.r. spectra were measured on a JEOL n s tetramethylsilane JNM-GX 400 spectrometer in chloroform-d, or dimethylsulfoxide-d, s o l ~ t i ~ using as internal standard. Mass spectra and high-resolution mass spectra were obtained with a JEOLJMS-SX 102spectrometer (ionization voltage 70 eV, accelerating voltage 10kV,ionization current 300pA) using a direct inlet system.
K. Tanaka et al.
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674
Isolation of metabolites (M1 and M2) Incubation was terminated by cooling to 0°C and adding 300 ml of ethyl acetate. Metabolic products were extracted three times with ethyl acetate and the organic solutions were combined, dried over anhydr. NazS04 and evaporated to dryness in wacuo. Metabolites M1 and M2 were isolated by repeated preparative t.1.c. using solvent systems (a) and (b), followed by recrystallization from methanol-ether. MI ( I I ) . Colourless needles (from methanol-ether), m.p. 161-165"C, [al0-22.6O (c=0.8, ethanol), U.V. Am,.nm (log&):285.4 (3.20) and 278, (3.15); i.r. v,,,cm-': 3320, 1710, 1589, 1460. 'H- and "CN.m.r.: tables 1 and 2. Mass spectrum m/z: 266 (M'), 249 (M+-OH), 235 (M+-CH,OH), 217 (M+CHzOH-H,O), 187, 137, High-resolution mass spectrum: found 2661516, calcd for Cl,H,,04 (M') 2661516; found 2491493, calcd for Cl,H2,03 2491493; found 235.1357, calcd for C14Hl,03 235.1334; found 217.1224, calcd for C,,H1,Oz 2171229; found 187.1100, calcd for C13Hls0 1871123; found 137.1331, calcd for CloH17 1371331. MZ ( I l l ) . Colourless needles (from methanol-ether), m.p. 145-146"C, [a],-265" (c=O.7, ethanol), u.v.A,,nm(log~): 2868(3.20)and278,,(3.15); i.r. v,,,cm-': 3322,1735,1589,1460,766. 'H-and 13CN.m.r.: tables 1 and 2. Mass spectrum m / z : 284 (M'), 286 (isotope peak), 267 (M+-OH), 269 (isotope peak), 251 (M+-CH3-H20),253 (isotope peak), 235 (M+-CHzCland M+-CH3-20H), 217,187. Highresolution mass spectrum: found 284.1 183, calcd for Cl,Hz103Cl (M') 2841 179; found 267.1143, calcd for CISH,,OzC1 2671 152; found 251-0854, calcd for C14H160zC1251-0839; found 2351372, calcd for C14Hl,03 235.1334; found 235.0934, calcd for C1,H160CI 235.0889; found 217.1218, calcd for C14H1702217.1229; found 1871125, calcd for CI3H,,O 187.1123.
Results ' H - and 13C-N.m.r. spectra of illudin S The 'H- and13C-n.m.r. spectra of illudin sesquiterpenoids have been studied in detail by Peter et al. (1982), who made assignments of all the 'H- and 13C-signalsin illudin S except those of the cyclopropane methylene protons. We have re-examined the n.m.r. spectra of illudin S by the use of two-dimensional (2D) n.m.r. techniques and differential nuclear Overhauser effect (NOE) experiments, and found that the previous assignments of the cyclopropane carbons must be revised (see figure 2). Two sets of 'H- and 3C-signalsdue to the cyclopropane methylene groups could readily be recognized at 13~0.42(ddd, J = 9*7,6-7,4.1Hz) and 1.12 (ddd, J = 9.9,6*1, 41 Hz) and ~ 5 ~ 8 . 7 (t)6 and at 6,084 (ddd, J=9*9,67,49 Hz), and 096 (ddd, J =9.7, 6-1, 4.9Hz) and 6, 6.04 (t) by means of 'H-'H and 'H-13C shift correlation spectroscopy (COSY) (Bax1982). Next, irradiation of the tert.-methyl at 61.37 (10H3) enhanced the signal intensity of the proton at 60.84 (12-H), while irradiation of the vinyl methyl at 61-69 (13-H3) increased the signal intensities of the protons at ~ 5 ~ 0 . 4(11-H), 2 0-96(12-H), and 4.72 (6-H), as shown in figure 2. It follows that the 'H-signals at 60.42 and 1-12and at 60.84 and 096 should be assigned to l l - H 2 and 12-H,, respectively (tablel), and hence the "C-signals at 68.76 and 6-04to 11-C and 12-C, respectively. Other 13C-signalswere analysed by the use of 'H-13C COSY and 'H-13C long-range COSY and the results are given in table 2.
'
Structures of metabolites T.1.c. analysis of the ethyl acetate extract of the incubation mixture showed two metabolite spots (Ml: & 033 and 0.25; M2: R,0.75 and 0.43; developing solvent (a) and (b), respectively). These new spots were ascribed to the metabolites of illudin S, since they were not observed on t.1.c. of the incubation mixture in the absence of the substrate or cofactor NADP. Furthermore, the spots were not observed when a heated 9000g supernatant was used. The time-course studies showed that the increase of the amounts of M1 and M2 was roughly proportional to the decrease of illudin S and the reaction was completed in about 25 min (figure 3). M1 showed the molecular ion peak at mlz 266 in the mass spectrum and its molecular formula was determined to be C 5H2204from the high-resolution mass
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Metabolites of illudin S
15
a)
1
.c
675
'L 12
CJ
lo
6 13
12
12
Figure 2. 'H-N.m.r. spectrum of illudin S. (a) Normal 'H-n.m.r. spectrum in CDC1,; (b), NOE difference spectrum irradiated at 61-20; (c), NOE difference spectrum irradiated at 61-37; (d), NOE difference spectrum irradiated at 61.69.
676
I(. Tanaka et al.
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1.01
....,.. subc?&teO
5 10 20 30 60 Incubation time (min)
Figure 3. Time-course of formation of M1 and M2 by incubation of iliudin S with rat liver 9000g supernatant. Ethyl acetate extracts obtained from mixtures incubated for different times were applied to t.1.c. plates and developed with ethyl acetatebenzene (6 :4, v/v) as a developing solvent. The mapping of spots on a plate was measured by using Shimadzu CS-9OOO scanner, with a sample beam at 225 nm and zig-zag scanning.
spectrum. In the U.V. spectrum M1 exhibited absorption bands at 278 and 2854 nm (log E : 3.16 and 3-20) and in the i.r. spectrum at 1589 and 1460cm-' together with hydroxyl absorption (3320cm- '), indicating the presence of a benzene ring. The 'H-n.m.r. spectrum of M1 exhibited signals due to a tert.-methyl (61-0.5))two benzylic methyls (62-10and 2.19))a benzylic methylene (62.44 and 2.57, each l H , d, J = 16.2 Hz), a hydroxyl-bearing methylene (63.37, t, J=8.2 Hz), and a hydroxylbearing methine (64.55, s). It also showed signals at 62.73 and 3.37 (each 2H, tripletlike, J = 8.2 Hz), which could be ascribed to a hydroxyethyl grouping, while the characteristic cyclopropane protons in the starting substance illudin S had disappeared. These spectral data led to the conclusion that the structure of M1 may be represented by the formula I1 (see figure l),which would be formed by reduction at the enone group followed by elimination of the 2-hydroxyl group and nucleophilic cleavage of the cyclopropane ring. This was corroborated by the analysis of its 13Cn.m.r. spectrum with the aid of distortionless enhancement by polarization transfer (DEPT) (figure 4) (Benn and Gunther 1983), 'H-"C COSY (figure 5) and 'H-l3C long-range COSY (figure 6). As shown in figure 6, the carbon atoms corresponding to the signals at 6148.65 (C-1) and at 6142.07 (C-5) are correlated with the protons corresponding to the signals at 62.10 (lo-CH,) and at 62-19(13-H) and 2.44 and 2-57 (8-H2), respectively. Similarly, quaternary carbons at 6134.25 (C-3), 12604 (C-9), 12437 (C-4), 122.29 (C-2) and 47.58 (C-7) can be correlated with the protons indicated by arrows in the formula in figure 6. Some of the other significant 'H-13C long-range correlations observed are also illustrated by arrows.
Metabolites of illudin S
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.
.
.
. . , .
677
..
I
150
100
"
'
50
Figure 4. Complete decoupled 13C-n.m.r. and DEPT (distortionless enhancement by polarization transfer) spectra of M1 (11) in DMSO-d,. T h e multiplicities of carbon signals are indicated by s, d, t, and q.
On the basis of these findings, the structure of M1 was determined as represented by the formula I1 shown in figure 1. M2 exhibited the molecular ion peak at mlz 284 and the isotope ion peak at mlz 286 in the ratio of 3 :1 in the mass spectrum (figure 7), indicating the presence of a chlorine atom in the molecule and its molecular formula C,,H,lO,Cl was established from the high-resolution mass spectrum. The U.V. and i.r. spectra of M2 were almost identical with those of M1, except for the appearance of a strong i.r. absorption band at 766 cm- which was assignable to a C-Cl bond. The 'H- and l3C-n.m.r. spectra of M2, analysed with the aid of 'H--l3C COSY and 'H-13C long-range COSY, were also very similar to those of MI except for slight differences in the chemical shift values for 'H- and 13C-signals assignable to the 11- and 12-methylene groups (tables 1 and 2). From the above spectral data and the molecular formula, the structure of M2 was determined to be I11 (figure 1).
',
Discussion The present paper describes preliminary experiments of biotransformation of illudin S with rat 9000g supernatant. Our results show that illudin S was biotransformed into two cyclopropane ring-cleavage metabolites (M1 and M2). It is worth notins that M2 is the first example of an organic chlorometabolite isolated from a incubation mixture using a tissue homogenate from a higher animal (Neidleman and Geigert 1986).
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678
K.Tanaka et al.
.
. .. I
, . .
...
Figure 6. 'H-13C Long-range shift correlation spectrum of M1 (11) in DMSO-d,. Arrows show 'H-13C long-range correlation observed.
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.._W
0
J
K. Tanaka et al.
251
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235
too
150
200
I 267
284 ( M*)
250
Figure 7. Mass spectnun of M2 (111).
Two enzymic attack sites are possible in the molecule of illudin S to produce both M1 and M2. The one is a,fi-unsaturated carbonyl group, and the other is Bcyclopropyl alkanol group. When dihydroilludin S (Ichihara et 01. 1969), a'reduced compound at the C,-ketone of illudin S, was used as the substrate, the starting material was recovered unchanged and production of any metabolite was not observed. It is therefore considered that the reactions at the two attack sites do not occur independently in the metabolism of illudin S. The initial step may be hydrogenation at the a, fi-unsaturated carbonyl system to give a highly reactive intermediate (IV) (figure 1). Elimination of the 2-hydroxy group in this intermediate and the concerted nucleophilic cleavage of the cyclopropane ring would give M1 and M2 (Brady et al. 1968). However, at present it is uncertain whether the above dehydration process is an enzymically controlled reaction or a non-enzymic followup reaction. Furthermore, illudin S might be transformed to other metabolites such as more polar metabolites and/or metabolites bound to biomacromolecules, as only about 50-60% of the substrate could be accounted for by the two metabolites identified. The mechanism and identification of the enzyme system producing these metabolites, and the pharmacological activity of these metabolites, are currently under investigation.
References BAX,A., 1982, Two-dimensional N M R in Liquids (Holland: D. Reidel). BENN,R., and GI~NTHER, H., 1983, Modem pulse methods in high-resolution NMR spectroscopy. Angmante Chemie (international edition in English), 22, 350-380. BRADY,S. F., ILTON,M. A., and JOHNSON, W. S., 1968, A high stereoselective synthesis of transtrisubstituted olefinic bonds. Journal of the American Chemical Society, 90,2882-2889. FUKUDA,K., UEMATSU,T., HAMADA, A., AKIYA,S., KOMATSU, N., and OKUBO,A., 1975, The polysaccharidefrom Lampteromyces japonicus. Chemical Pharmaceutical Bulletin, 23, 1955-1959. ICHIHARA,A., SHrmHAMA, H., and MATSUMOTO, T., 1969, Dihydroilludin S, a new constituent from Lampteromyces japonicus. Tetrahedron Letters, 45, 3965-3968. ITANO, T., 1978, Illudin S; a possible new type inhibitor of RNA synthesis, and analysis of its action mechanism. Okayoma Igaku Zasshi, 91,453-460. KELNER,M. J . , MCMORRIS, T. C., BECK,W. T., ZAMORA, J. M., and TAETLE, R., 1987, Preclinical evaluation of illudin S as anticancer agents. Cancer Research, 47, 3186-3189.
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MATSUMOTO, T., SHIRAHAMA, H., ICHIHARA, A., FUKUOKA, Y., TAKAHASHI, Y., MORI,Y., and WATANABE, M., 1965, Structure of lampterol (illudin S). Tetrahedron, 21, 2671-2676. NAKANISHI, K., OHASHI, M., SUZUKI, N., TADA,M., YAMADA, Y., and INAGAKI, S., 1963 a, Isolation of lampterol from Lampteromyces japonicus (KAWAM.) SING. Yakugaku Zasshi, 83, 377-380. NAKANISHI, K., TADA,M., YAMADA, Y .,OHASHI, M., KOMATSU, N., and TERAKAWA, H., 1963 b, Isolation of lampterol, an antitumour substance from Lampterumyces japoninrc. Nature, 197, 292. NAKANIHSI, K., TADA,M., and YAMADA, Y., 1964, Stereochemistry of illudin-S (lampterol). Chemical Pharmaceutical Bulletin, 12, 856-857. NAKANISHI, K., OHASHI, M., TADA,M., and YAMADA, Y., 1965, Illudin S (lampterol). Tetrahedron, 21, 1231-1246. NEIDLEMAN, S . L., and GEIGERT, J., 1986, Bwhalogenation: Principles, Baric Roles and Applications
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(Chichester: Ellis Horwood).
PETER, A., BREDSHAW, W., HANSON, J. R., and SADLER, I. H., 1982, Studies in terpenoid biosynthesis. Part 26. Applicationsof 'H and "C n.m.r. spectroscopyto the biosynthesis of the illudin sesquiterpenoids. Journal of the Chemical Society Perkin Transactions, I , 10, 2445-2448. SHINOZAWA, K., TSUTSUI, K., and ODA,T., 1979, Enhancement of the effect of illudin S by including it into liposomes. Expm'entia, 35, 1102-1 103. SUCKLING, C. J., 1988, The cyclopropylgroup in studies of enzymemechanism and inhibition. Angmante Chemie (international edition in English), 27, 537-552. TADA, M., YAMADA, Y .,BHACCA, N. S., NAKANISHI, K., and OHASHI, M., 1964, Structure and reactions of illudin S (lampterol). Chemical Pharmaceutical Bulletin, 12, 853-855. TSUDA, R., KAWANO, M., and HARA,M., 1986, Phytohemagglutinin found in Lampterumycesjaponicus. Zgakukenkyu, 56,22-26.
1990, VOL. 20,
NO.
7, 671-681
Metabolism of illudin S, a toxic principle of Lampteromycesjaponicus, by rat liver. I. Isolation and identification of cyclopropane ring-cleavage metabolites
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K. TANAKAT, T. INOUET, S. KADOTAS and T. KIKUCHIS
t National Research Institute of Police Science, 6, Sanban-cho, Chiyoda-ku, Tokyo 102, Japan # Research Institute for Wakan-Yaku (Oriental Medicines), Toyama Medical and Pharmaceutical University, 2630, Sugitani, Toyama 930-01, Japan Received 14 September 1989; accepted 2 March 1990
1. Illudin S, a toxic principle of the basidiomycete Lmnpteromyces japonzcuc, was incubated with rat liver 9OOOg supernatant and its metabolites studied. 2. Two metabolites, M1 and M2, were isolated and identified as cyclopropane ringcleavage compounds by n.m.r., i.r. and mass spectral analyses. Moreover, M2 contained a chlorine atom. 3. On the basis of detailed analyses of the 2D n.m.r. spectra and differential nuclear Overhauser effect experiments, the previous assignments of the cyclopropane carbons of illudin S were revised.
Introduction The basidiomycete Lampteromyces japonicus (Japanese name: Tsukiyo-Take) is distinctive in being bioluminescent and is one of the most notorious poisonous mushrooms in Japan. There have been reports of severe illness caused by this mushroom, because occasionally it is eaten by mistake due to its resemblance to edible mushrooms. Chemical investigation of the constituents of this mushroom have been undertaken by several groups of authors (Fukuda et al. 1975, Tsuda et al. 1986) and the isolation of a toxic substance, illudin S (I), having a novel structure with a cyclopropane ring (figure l),has been reported (Nakanishi et al. 1963a, Tada et al. 1964, Nakanishi et al. 1964,1965,Matsumoto et al. 1965). In addition, illudin S has been reported to have antibacterial and antiturnour properties (Itano 1978, Shinozawa et al. 1979, Kelner et al. 1987). Cyclopropane-ring-containing compounds are currently of increasing interest and are used in enzymology, toxicology, etc., and the bioorganic chemistry of cyclopropanes has been explored extensively. It has been demonstrated that in many cases ring-opened metabolites are produced by enzymic oxidation (Suckling 1988). However, no report on the metabolism of illudin. S has yet been published, although knowledge of the metabolic pathways of illudin S in animals and of any biological activities of the metabolites, would be of considerable interest. This paper therefore describes the isolation and identification of illudin S metabolites formed by rat liver 9000g supernatant.
Materials and methods Chicals Illudin S was isolated according to published methods (Nakanishi et al. 1963 a, b, 1964, 1965), from Lampterornyces japomcuc collected in Nagano prefecture in October 1987. 0049-8254/90 $3.00 0 1990 Taylor & Francis Ltd.
K. Tanaka et al.
672
illudin S ( I )
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1
I
OH
M1 (11)
M2(III)
Figure 1. Proposed pathways of metabolism of illudin S by rat liver 9OOOg supernatant, Postulated intermediate shown in parentheses. Illudin S (I): m.p. 125-126"C, [&+ 15lo(c=0.1, methanol), U.V. A M x n m (log 8): 2340 (409), 3180 (3.51);i.r.vmaxcm-': 3408,1693,1659,1600. 'H-and13C-N.m.r.:tables1 and2.Massspectrumm/a: 264 (M'), 233,216,201,189,173. Identity was confirmed by comparing the physical and spectral data with those published. NADP was purchased from Sigma Chemical Co. Ltd (St Louis,MO, USA). All other chemicals and reagents were of the highest grade. Preparation of 9000g supernatant Wistar male rats weighing 150-200 g were obtained from Nippon Bio-Supp. Center (Tokyo, Japan), and allowed free access to standard laboratory chow (MF, Oriental Yeast Co., Tokyo, Japan) and tap water. Rats were killed by decapitation, and livers were quickly removed and chilled in isotonic KCI. A homogenate was prepared by hand, using a glass homogenizer with a Teflon pestle using 2 ml of isotonic KCI per 1 g of liver. A 9OOOg supernatant fraction was prepared by centrifugation of the homogenate for 20min in a Marusan 50-B refrigerated centrifuge. All procedures were carried out at 0 4 ° C . The 9000g supernatant was used fresh on its day of preparation. Incubation A solution of illudin S ( 2 5 0 p o l ) in ethanol ( 2 5 0 ~ 1was ) added to a mixture of NADP (SOpmol), MgCI2(5mmol),and50mlof0.4~phosphatebuffer(pH7.4).Tothismixturewasadded 100mlof9000g supernatant and water to 250 ml. The mixture was incubated for 0.5 h at 37"C, with shaking. For a timecourse study, incubation mixtures consisted of 0 4 m l of 9000gsupernatant fraction, 2.5 pmol of illudin S,
Metabolites of illudin S
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Table 1. 'H-N.m.r. data for illudin S, metabolites M1 and M2 (ppm from tetramethylsilane) Compound Illudin S'
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Proton 472 s 645 s
6-H 8-H 8-H 10-H 11-H 11-H 12-H 12-H 13-H 14-H 15-H 15-H
Mlb 455 s
1-37s 0.42 ddd (97,67,41) 1.12ddd(9.9,6.1,4.1) 0.84 ddd (9.9, 67, 49) @96ddd(97,6*1,49) 1.69 s 1.20s 3.47d(ll) 3.55 d(l1)
MZb
2.44 d (16.2) 2.57 d (162) 210s 273 t (82)
4.57 s 245 d (159) 2-59d (15.9) 211 8 3-02t (98)
3.37 t (82)
3-59t (98)
219s 1-05s 3-01d(10.4) 3*07d(10.4)
221 8 1.04s 3.01 d(10.4) 3.07 d (10.4)
Spectra were measured in CDCI,' and DMSO-d,b solutions. Values in parentheses are coupling constants (Hz). Table 2.
',C-N.m.r. data for illudin S, metabolites M1 and M2 Compound ~
Carbon atom 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Illudin S' 20050(s) 7615 ( 8 ) 3190 (s) 13504( 8 ) 13852 (s) 7484 (d) 551O(s) 141.57(d) 136.06 (s) 2480 (9) 876 (t) 6.04 (t) 1416(q) 15.83 (9) 68.94 (t)
Mlb 148.65 ( 8 ) 122.29 (s) 13425 ( 8 ) 124.37 (s) 14207(s) 7689 (d) 47.58 ( 8 ) 37.60 (t) 12604(s) 11-97(9) 33-44(t) 6Q16(t) 1455 (4) 1689(9) 6767 (t)
M2b 148.82 ( 8 ) 12244 ( 8 ) 133*34(s) 12450 (s) 142-38(s) 7681 (d) 47.58 ( 8 ) 3759 (t) 127-05(s) 11.91 (9) 33*00(t) 42-84(t) 14.46 (9) 1861 (9) 67-58(t)
Spectra were measured in CDCI,' and DMSO-d,'solutions. The multiplicities of carbon signals are indicated as s, d, t, and q. 1pmol of NADP, 100pnol of MgCI,, 400pnol of phosphate buffer (pH 7.4), and water to a final volume of 5mI. Analytical methodc T.1.c. was carried out on Merck Kieselgel60 F ,,, (0.25 mm) platea using (a) ethyl acetatebenzene (6 :4, v/v) or (b) chloroform-methanol(9 :1, v/v) as developing solvents. Spots were detected under U.V. light (254nm) or from coloration by spraying with anisaldehyd-ulphuric acid-ethanol(1: 1 :19,by vol.) reagent and heating at 120°C for 5min. T.1.c. densitometrywas carried out on a Shmadzu CS-9OOO scanner, with the sample beam at 225 nm and zig-zag scanning. Melting points were determined with a Kofler-type apparatus and are uncorrected. U.V.spectra were taken in ethanol solutions and i.r. spectra in KBr discs. N.m.r. spectra were measured on a JEOL n s tetramethylsilane JNM-GX 400 spectrometer in chloroform-d, or dimethylsulfoxide-d, s o l ~ t i ~ using as internal standard. Mass spectra and high-resolution mass spectra were obtained with a JEOLJMS-SX 102spectrometer (ionization voltage 70 eV, accelerating voltage 10kV,ionization current 300pA) using a direct inlet system.
K. Tanaka et al.
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Isolation of metabolites (M1 and M2) Incubation was terminated by cooling to 0°C and adding 300 ml of ethyl acetate. Metabolic products were extracted three times with ethyl acetate and the organic solutions were combined, dried over anhydr. NazS04 and evaporated to dryness in wacuo. Metabolites M1 and M2 were isolated by repeated preparative t.1.c. using solvent systems (a) and (b), followed by recrystallization from methanol-ether. MI ( I I ) . Colourless needles (from methanol-ether), m.p. 161-165"C, [al0-22.6O (c=0.8, ethanol), U.V. Am,.nm (log&):285.4 (3.20) and 278, (3.15); i.r. v,,,cm-': 3320, 1710, 1589, 1460. 'H- and "CN.m.r.: tables 1 and 2. Mass spectrum m/z: 266 (M'), 249 (M+-OH), 235 (M+-CH,OH), 217 (M+CHzOH-H,O), 187, 137, High-resolution mass spectrum: found 2661516, calcd for Cl,H,,04 (M') 2661516; found 2491493, calcd for Cl,H2,03 2491493; found 235.1357, calcd for C14Hl,03 235.1334; found 217.1224, calcd for C,,H1,Oz 2171229; found 187.1100, calcd for C13Hls0 1871123; found 137.1331, calcd for CloH17 1371331. MZ ( I l l ) . Colourless needles (from methanol-ether), m.p. 145-146"C, [a],-265" (c=O.7, ethanol), u.v.A,,nm(log~): 2868(3.20)and278,,(3.15); i.r. v,,,cm-': 3322,1735,1589,1460,766. 'H-and 13CN.m.r.: tables 1 and 2. Mass spectrum m / z : 284 (M'), 286 (isotope peak), 267 (M+-OH), 269 (isotope peak), 251 (M+-CH3-H20),253 (isotope peak), 235 (M+-CHzCland M+-CH3-20H), 217,187. Highresolution mass spectrum: found 284.1 183, calcd for Cl,Hz103Cl (M') 2841 179; found 267.1143, calcd for CISH,,OzC1 2671 152; found 251-0854, calcd for C14H160zC1251-0839; found 2351372, calcd for C14Hl,03 235.1334; found 235.0934, calcd for C1,H160CI 235.0889; found 217.1218, calcd for C14H1702217.1229; found 1871125, calcd for CI3H,,O 187.1123.
Results ' H - and 13C-N.m.r. spectra of illudin S The 'H- and13C-n.m.r. spectra of illudin sesquiterpenoids have been studied in detail by Peter et al. (1982), who made assignments of all the 'H- and 13C-signalsin illudin S except those of the cyclopropane methylene protons. We have re-examined the n.m.r. spectra of illudin S by the use of two-dimensional (2D) n.m.r. techniques and differential nuclear Overhauser effect (NOE) experiments, and found that the previous assignments of the cyclopropane carbons must be revised (see figure 2). Two sets of 'H- and 3C-signalsdue to the cyclopropane methylene groups could readily be recognized at 13~0.42(ddd, J = 9*7,6-7,4.1Hz) and 1.12 (ddd, J = 9.9,6*1, 41 Hz) and ~ 5 ~ 8 . 7 (t)6 and at 6,084 (ddd, J=9*9,67,49 Hz), and 096 (ddd, J =9.7, 6-1, 4.9Hz) and 6, 6.04 (t) by means of 'H-'H and 'H-13C shift correlation spectroscopy (COSY) (Bax1982). Next, irradiation of the tert.-methyl at 61.37 (10H3) enhanced the signal intensity of the proton at 60.84 (12-H), while irradiation of the vinyl methyl at 61-69 (13-H3) increased the signal intensities of the protons at ~ 5 ~ 0 . 4(11-H), 2 0-96(12-H), and 4.72 (6-H), as shown in figure 2. It follows that the 'H-signals at 60.42 and 1-12and at 60.84 and 096 should be assigned to l l - H 2 and 12-H,, respectively (tablel), and hence the "C-signals at 68.76 and 6-04to 11-C and 12-C, respectively. Other 13C-signalswere analysed by the use of 'H-13C COSY and 'H-13C long-range COSY and the results are given in table 2.
'
Structures of metabolites T.1.c. analysis of the ethyl acetate extract of the incubation mixture showed two metabolite spots (Ml: & 033 and 0.25; M2: R,0.75 and 0.43; developing solvent (a) and (b), respectively). These new spots were ascribed to the metabolites of illudin S, since they were not observed on t.1.c. of the incubation mixture in the absence of the substrate or cofactor NADP. Furthermore, the spots were not observed when a heated 9000g supernatant was used. The time-course studies showed that the increase of the amounts of M1 and M2 was roughly proportional to the decrease of illudin S and the reaction was completed in about 25 min (figure 3). M1 showed the molecular ion peak at mlz 266 in the mass spectrum and its molecular formula was determined to be C 5H2204from the high-resolution mass
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Metabolites of illudin S
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a)
1
.c
675
'L 12
CJ
lo
6 13
12
12
Figure 2. 'H-N.m.r. spectrum of illudin S. (a) Normal 'H-n.m.r. spectrum in CDC1,; (b), NOE difference spectrum irradiated at 61-20; (c), NOE difference spectrum irradiated at 61-37; (d), NOE difference spectrum irradiated at 61.69.
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1.01
....,.. subc?&teO
5 10 20 30 60 Incubation time (min)
Figure 3. Time-course of formation of M1 and M2 by incubation of iliudin S with rat liver 9000g supernatant. Ethyl acetate extracts obtained from mixtures incubated for different times were applied to t.1.c. plates and developed with ethyl acetatebenzene (6 :4, v/v) as a developing solvent. The mapping of spots on a plate was measured by using Shimadzu CS-9OOO scanner, with a sample beam at 225 nm and zig-zag scanning.
spectrum. In the U.V. spectrum M1 exhibited absorption bands at 278 and 2854 nm (log E : 3.16 and 3-20) and in the i.r. spectrum at 1589 and 1460cm-' together with hydroxyl absorption (3320cm- '), indicating the presence of a benzene ring. The 'H-n.m.r. spectrum of M1 exhibited signals due to a tert.-methyl (61-0.5))two benzylic methyls (62-10and 2.19))a benzylic methylene (62.44 and 2.57, each l H , d, J = 16.2 Hz), a hydroxyl-bearing methylene (63.37, t, J=8.2 Hz), and a hydroxylbearing methine (64.55, s). It also showed signals at 62.73 and 3.37 (each 2H, tripletlike, J = 8.2 Hz), which could be ascribed to a hydroxyethyl grouping, while the characteristic cyclopropane protons in the starting substance illudin S had disappeared. These spectral data led to the conclusion that the structure of M1 may be represented by the formula I1 (see figure l),which would be formed by reduction at the enone group followed by elimination of the 2-hydroxyl group and nucleophilic cleavage of the cyclopropane ring. This was corroborated by the analysis of its 13Cn.m.r. spectrum with the aid of distortionless enhancement by polarization transfer (DEPT) (figure 4) (Benn and Gunther 1983), 'H-"C COSY (figure 5) and 'H-l3C long-range COSY (figure 6). As shown in figure 6, the carbon atoms corresponding to the signals at 6148.65 (C-1) and at 6142.07 (C-5) are correlated with the protons corresponding to the signals at 62.10 (lo-CH,) and at 62-19(13-H) and 2.44 and 2-57 (8-H2), respectively. Similarly, quaternary carbons at 6134.25 (C-3), 12604 (C-9), 12437 (C-4), 122.29 (C-2) and 47.58 (C-7) can be correlated with the protons indicated by arrows in the formula in figure 6. Some of the other significant 'H-13C long-range correlations observed are also illustrated by arrows.
Metabolites of illudin S
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.
.
.
. . , .
677
..
I
150
100
"
'
50
Figure 4. Complete decoupled 13C-n.m.r. and DEPT (distortionless enhancement by polarization transfer) spectra of M1 (11) in DMSO-d,. T h e multiplicities of carbon signals are indicated by s, d, t, and q.
On the basis of these findings, the structure of M1 was determined as represented by the formula I1 shown in figure 1. M2 exhibited the molecular ion peak at mlz 284 and the isotope ion peak at mlz 286 in the ratio of 3 :1 in the mass spectrum (figure 7), indicating the presence of a chlorine atom in the molecule and its molecular formula C,,H,lO,Cl was established from the high-resolution mass spectrum. The U.V. and i.r. spectra of M2 were almost identical with those of M1, except for the appearance of a strong i.r. absorption band at 766 cm- which was assignable to a C-Cl bond. The 'H- and l3C-n.m.r. spectra of M2, analysed with the aid of 'H--l3C COSY and 'H-13C long-range COSY, were also very similar to those of MI except for slight differences in the chemical shift values for 'H- and 13C-signals assignable to the 11- and 12-methylene groups (tables 1 and 2). From the above spectral data and the molecular formula, the structure of M2 was determined to be I11 (figure 1).
',
Discussion The present paper describes preliminary experiments of biotransformation of illudin S with rat 9000g supernatant. Our results show that illudin S was biotransformed into two cyclopropane ring-cleavage metabolites (M1 and M2). It is worth notins that M2 is the first example of an organic chlorometabolite isolated from a incubation mixture using a tissue homogenate from a higher animal (Neidleman and Geigert 1986).
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K.Tanaka et al.
.
. .. I
, . .
...
Figure 6. 'H-13C Long-range shift correlation spectrum of M1 (11) in DMSO-d,. Arrows show 'H-13C long-range correlation observed.
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251
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235
too
150
200
I 267
284 ( M*)
250
Figure 7. Mass spectnun of M2 (111).
Two enzymic attack sites are possible in the molecule of illudin S to produce both M1 and M2. The one is a,fi-unsaturated carbonyl group, and the other is Bcyclopropyl alkanol group. When dihydroilludin S (Ichihara et 01. 1969), a'reduced compound at the C,-ketone of illudin S, was used as the substrate, the starting material was recovered unchanged and production of any metabolite was not observed. It is therefore considered that the reactions at the two attack sites do not occur independently in the metabolism of illudin S. The initial step may be hydrogenation at the a, fi-unsaturated carbonyl system to give a highly reactive intermediate (IV) (figure 1). Elimination of the 2-hydroxy group in this intermediate and the concerted nucleophilic cleavage of the cyclopropane ring would give M1 and M2 (Brady et al. 1968). However, at present it is uncertain whether the above dehydration process is an enzymically controlled reaction or a non-enzymic followup reaction. Furthermore, illudin S might be transformed to other metabolites such as more polar metabolites and/or metabolites bound to biomacromolecules, as only about 50-60% of the substrate could be accounted for by the two metabolites identified. The mechanism and identification of the enzyme system producing these metabolites, and the pharmacological activity of these metabolites, are currently under investigation.
References BAX,A., 1982, Two-dimensional N M R in Liquids (Holland: D. Reidel). BENN,R., and GI~NTHER, H., 1983, Modem pulse methods in high-resolution NMR spectroscopy. Angmante Chemie (international edition in English), 22, 350-380. BRADY,S. F., ILTON,M. A., and JOHNSON, W. S., 1968, A high stereoselective synthesis of transtrisubstituted olefinic bonds. Journal of the American Chemical Society, 90,2882-2889. FUKUDA,K., UEMATSU,T., HAMADA, A., AKIYA,S., KOMATSU, N., and OKUBO,A., 1975, The polysaccharidefrom Lampteromyces japonicus. Chemical Pharmaceutical Bulletin, 23, 1955-1959. ICHIHARA,A., SHrmHAMA, H., and MATSUMOTO, T., 1969, Dihydroilludin S, a new constituent from Lampteromyces japonicus. Tetrahedron Letters, 45, 3965-3968. ITANO, T., 1978, Illudin S; a possible new type inhibitor of RNA synthesis, and analysis of its action mechanism. Okayoma Igaku Zasshi, 91,453-460. KELNER,M. J . , MCMORRIS, T. C., BECK,W. T., ZAMORA, J. M., and TAETLE, R., 1987, Preclinical evaluation of illudin S as anticancer agents. Cancer Research, 47, 3186-3189.
Metabolites of illudin S
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MATSUMOTO, T., SHIRAHAMA, H., ICHIHARA, A., FUKUOKA, Y., TAKAHASHI, Y., MORI,Y., and WATANABE, M., 1965, Structure of lampterol (illudin S). Tetrahedron, 21, 2671-2676. NAKANISHI, K., OHASHI, M., SUZUKI, N., TADA,M., YAMADA, Y., and INAGAKI, S., 1963 a, Isolation of lampterol from Lampteromyces japonicus (KAWAM.) SING. Yakugaku Zasshi, 83, 377-380. NAKANISHI, K., TADA,M., YAMADA, Y .,OHASHI, M., KOMATSU, N., and TERAKAWA, H., 1963 b, Isolation of lampterol, an antitumour substance from Lampterumyces japoninrc. Nature, 197, 292. NAKANIHSI, K., TADA,M., and YAMADA, Y., 1964, Stereochemistry of illudin-S (lampterol). Chemical Pharmaceutical Bulletin, 12, 856-857. NAKANISHI, K., OHASHI, M., TADA,M., and YAMADA, Y., 1965, Illudin S (lampterol). Tetrahedron, 21, 1231-1246. NEIDLEMAN, S . L., and GEIGERT, J., 1986, Bwhalogenation: Principles, Baric Roles and Applications
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(Chichester: Ellis Horwood).
PETER, A., BREDSHAW, W., HANSON, J. R., and SADLER, I. H., 1982, Studies in terpenoid biosynthesis. Part 26. Applicationsof 'H and "C n.m.r. spectroscopyto the biosynthesis of the illudin sesquiterpenoids. Journal of the Chemical Society Perkin Transactions, I , 10, 2445-2448. SHINOZAWA, K., TSUTSUI, K., and ODA,T., 1979, Enhancement of the effect of illudin S by including it into liposomes. Expm'entia, 35, 1102-1 103. SUCKLING, C. J., 1988, The cyclopropylgroup in studies of enzymemechanism and inhibition. Angmante Chemie (international edition in English), 27, 537-552. TADA, M., YAMADA, Y .,BHACCA, N. S., NAKANISHI, K., and OHASHI, M., 1964, Structure and reactions of illudin S (lampterol). Chemical Pharmaceutical Bulletin, 12, 853-855. TSUDA, R., KAWANO, M., and HARA,M., 1986, Phytohemagglutinin found in Lampterumycesjaponicus. Zgakukenkyu, 56,22-26.
